The Rotor-Gene Multiplex PCR Kit is designed for use with theRotor-Gene Q and other Rotor-Gene cyclers, providing ultrafast, highly reliable quantification in multiplex, real-time PCR and two-step RT-PCR using sequence-specific probes. Depending on the cycler configuration, up to 4 cDNA or gDNA targets (e.g., 1 control gene and 3 target genes) can be quantified simultaneously in the same tube. Outstanding performance is achieved through the combination of a specially optimized master mix and the unique Rotor-Gene cycler. For convenience, the master mix can be stored at 2–8°C.

Duplex, real-time two-step RT-PCR was carried out on the Rotor-Gene Q using the Rotor-Gene Multiplex PCR Kit and self-designed TaqMan assays for [A] IL8 (interleukin 8) and [B] ACTB (β-actin). Analysis of tenfold dilutions of leukocyte cDNA template from 100 ng to 1 pg provided high PCR efficiencies of around 95%. [C] The CT values were comparable with those achieved in control singleplex reactions, demonstrating the reliability of the duplex assay.|Tenfold dilutions of human leukocyte cDNA (10 ng to 10 pg) were used as template in 4-plex, real-time PCR. Reactions were run in triplicate using either the Rotor-Gene Q and Rotor-Gene Multiplex PCR Kit or an instrument and kit from Supplier S. [A] Targets amplified, and reporter dyes of corresponding TaqMan probes. [B] CT values obtained for all 4 targets (instrument and kit from Supplier S did not successfully detect IFNG; N.D.). Lower CT values on the Rotor-Gene Q demonstrate detection with greater sensitivity. [C] Amplification plots for TNF using the Rotor-Gene Multiplex PCR Kit. [D] Amplification plots for TNF using a kit from Supplier S. [E] Amplification plots for IFNG using the Rotor-Gene Multiplex PCR Kit. [F] Amplification plots for IFNG using a kit from Supplier S.|[A] Q-Bond in Rotor-Gene Multiplex PCR Master Mix increases the affinity of DNA polymerase for short single-stranded DNA, reducing primer annealing time to a few seconds. In addition, the unique buffer composition supports the melting of DNA, reducing denaturation and extension times. [B] Without Q-Bond, the primer and polymerase bind sequentially to the template, increasing primer annealing time.|Rotor-Gene Multiplex PCR Kits provide a simple procedure for quantitative, multiplex, real-time PCR. In contrast to current methods, the kits eliminate the need for optimization of the concentrations of primers, Mg2+, and Taq DNA polymerase, even for difficult assays (e.g., assays in which the copy number of the target gene is much smaller than that for the reference gene).|[A] NH4+ ions prevent nonspecific primers from annealing to the template. [B] Synthetic Factor MP, an innovative PCR additive, increases the local concentration of primers at the template. Together with K+ and other cations, Factor MP stabilizes specifically bound primers, allowing efficient primer extension by HotStarTaq Plus DNA Polymerase.|

With the Rotor-Gene Multiplex PCR Kit, up to 4 cDNA targets can be simultaneously and rapidly quantified in the same tube, increasing throughput and saving precious sample material (see figures "Reliable multiplex analysis" and "Reliable duplex analysis"). Genes of different expression levels are all amplified in the same tube with the same high efficiency, enabling reliable relative quantification of gene expression.

Principle

Amplifying reference and target genes in the same reaction instead of in separate reactions increases the reliability of gene quantification by minimizing handling errors. The Rotor-Gene Multiplex PCR Kit enables reliable multiplex real-time PCR quantification of cDNA targets on the Rotor-Gene Q without the need for optimization of reaction and cycling conditions (see flowchart "QIAGEN multiplex kits"). Highly specific amplification is assured through a balanced combination of K+ and NH4+ ions, which promote specific primer annealing and enable high PCR specificity and sensitivity, while synthetic Factor MP, an innovative PCR additive specially developed for challenging multiplex PCR applications, allows different amplicons in the same reaction to all be amplified with the same high efficiency (see figure "Unique PCR buffer").

Fast cycling without compromising performance is achieved using Q-Bond, a novel PCR additive that considerably shortens cycler run times (see figure "Fast primer annealing"). In addition, the highly stringent hot-start enzyme HotStarTaq Plus DNA Polymerase is rapidly activated at the start of PCR by a brief 5-minute incubation at 95ºC.

Components of 2x Rotor-Gene Multiplex PCR Kit*

Component

Features

Benefits

HotStarTaq Plus DNA Polymerase

5 min activation at 95ºC

Set up of qPCR reactions at room temperature

Rotor-Gene Multiplex PCR Buffer

Balanced combination of NH4+ and K+ ions

Specific primer annealing ensures reliable qPCR results

Synthetic Factor MP

Reliable multiplexing analysis of up to 4 targets in the same tube

Unique Q-Bond additive

Faster PCR run times enable faster results and more reactions per day

* Also contains dNTP mix (dATP, dCTP, dGTP, dTTP).

Procedure

A ready-to-use master mix eliminates the need for optimization of reaction and cycling conditions. Simply add template cDNA and primer-probe sets to the master mix and program the cycler. The handbook supplied with the kit lists recommended dyes and contains a single protocol for all multiplex qPCR assays.

The Rotor-Gene Multiplex PCR Kit is optimized for fast, real-time PCR and two-step RT-PCR analysis using sequence-specific probes on the Rotor-Gene Q. It is also compatible with the Rotor-Gene 3000 and the Rotor-Gene 6000. Up to 4 cDNA or gDNA targets can be simultaneously and rapidly quantified in the same tube, increasing throughput and saving precious sample material.